Dendritic cell line

ABSTRACT

The invention relates to plasmacytoid dendritic human cell lines and methods for producing the cell lines. More specifically, the invention relates to the plasmacytoid dendritic human cell line called GEN2.2, which is deposited in the CNCM under number CNCM 1-2938 and the plasmacytoid dendritic human cell line called GEN3, which is deposited in the CNCM under number I-3110. The use of cells from the cell lines is also disclosed.

The present invention relates to human plasmacytoid dendritic cell (pDC)lines, and to the methods for obtaining and for culturing these cells.The invention also relates to the uses of these cells and topharmaceutical compositions comprising these human plasmacytoiddendritic cells (pDCs).

Dendritic cells are key protagonists in the immune response: they areresponsible for capturing antigens and processing them for the purposeof presenting them to T lymphocytes. Various dendritic cell precursorshave been isolated from the blood; they express HLA-DR and CD4, in theabsence of any other line-specific marker. Among these precursors, themost well characterized are of myeloid origin, expressing CD11c, CD13and CD33 molecules, and differentiate (inter alia) into Langerhanscells; these cells are also called DC1. Another population ofprecursors, CD11c−, characterized by a very high level of expression ofthe IL3 receptor (CD123) has, moreover, been identified, and is itselfalso capable of differentiating into mature dendritic cells under theeffect of IL3, or in the presence of a virus; they are called DC2, orplasmacytoid dendritic cells (pDCs).

pDCs were identified specifically from tonsils in 1997 by G. Grouard etal. (G. Grouard et al., J. Exp. Med., 1997;185:1101-1111); they havealso been described in the blood (O'Doherty U. et al., Immunology,1994;82:487-493; Robinson S P. et al., Eur. J. Immunol.,1999;29:2769-2778), in the lymph nodes (Cella M. et al., Nat. Med.,1999;5:919-923) and in the thymus (Res P C. et al., Blood.,1999;94:2647-2657; Bendriss-Vermare N. et al., J. Clin. Invest.,2001;107:835-844). These cells are characterized by theirplasmacytoid-type morphology and their phenotype. pDCs express CD4,HLA-DR and CD45RA molecules, and lack the myeloid-type markers CD11c andCD13 (Cella M. et al., Nat. Med., 1999;5:919-923) or line-specificmarkers such as CD3, CD14 and CD19, although expression of CD2, CD5 orCD7 has sometimes been observed (Cella M., et al., Nat. Med.,1999;5:919-923; Res P C. et al., Blood., 1999;94:2647-2657). Morerecently, it has been possible to identify the lectin BDCA2 specificallyexpressed by pDCs; BDCA4 is found on pDCs, but is also present onmonocyte-derived DCs (Dzionek A. et al., J. Immunol., 2000;165:6037-6046and Human Immunology, Vol. 63, 2002, pages 1133-1148). An argument infavor of these cells belonging to the lymphoid line is the fact thatthey express mRNAs encoding the chains preTalpha (Res P C., et al.,Blood., 1999;94:2647-2657), lambda like 14.1 and SpiB (Bendriss-VermareN., et al., J. Clin. Invest., 2001;107:835-844). These cells verystrongly express the IL-3 receptor and weakly express the GM-CSFreceptor (Cella M. et al., Nat. Med., 1999;5:919-923; Rissoan M C. etal., Science., 1999;283:1183-1186) and these two cytokines promote thesurvival of pDCs (Grouard G. et al., J. Exp. Med., 1997;185:1101-1111;Kohrgruber N. et al., J. Immunol., 1999;163:3250-3259; Robinson S P. etal., Eur J. Immunol., 1999;29:2769-2778), which otherwise die veryrapidly in vitro. The costimulatory molecules CD80 and CD86 are absentor weakly expressed (Grouard G. et al., J. Exp. Med.,1997;185:1101-1111) and, at the immature stage, these cells areincapable of activating T lymphocytes (Kohrgruber N. et al., J.Immunol., 1999;163:3250-3259). On the other hand, in the presence ofIL-3, of CD40L or of a virus, mature pDCs strongly express secondaryantigen-presenting molecules (CD40, CD80, CD86 and HLA-DR) and thenbecome as powerful as DCs of myeloid origin for activating allogenicT-cell proliferation (Kohrgruber N. et al., J. Immunol.,1999;163:3250-3259; Grabbe S. et al., Immunol Today., 2000;21:431-433;Kadowaki N. et al., J. Exp. Med., 2000;192:219-226). According to thestimulus responsible for their maturation (IL3 or virus), pDCs willpolarize the response of the naive T lymphocytes that they activate, ina more or less strict manner, toward a Th1 or Th2 profile (Rissoan M C.et al., Science, 1999;283:1183-1186; Cella M. et al., Nat. Immunol.,2000;l:305-310; Kadowaki N. et al., J. Exp. Med., 2000;192:219-226).

Moreover, pDCs at the immature stage are responsible for the secretionof IFN-alpha detected in response to viruses (Cella M., et al., Nat.Med., 1999;5:919-923; Siegal F P et al., Science, 1999;284:1835-1837):they correspond to the “interferon producing cells” described in the1980s. In addition, they are capable of responding to bacterial DNAs andto products of viral origin that they recognize since they express TLR7(Ito T. et al., J. Exp. Med., 2002;195:1507-1512) and TLR9 (Bauer S. etal., Proc. Natl. Acad. Sci. USA, 2001;98:9237-9242) molecules. NormalpDCs therefore lie at the forefront of innate and adaptive immuneresponses, and could have a very important role in the initiation ofantiviral responses.

Plasmacytoid dendritic cells (pDCs) open up new therapeutic perspectivessince these cells are key protagonists in the immune response. Dendriticcells thus play an essential role in immune defenses with respect toinfectious agents (bacteria, viruses, parasites), in immune defenseswith respect to cancers, in allergic processes, in autoimmune responses,in the induction of tolerance and in transplant immunology. Dendriticcells are thus used in various therapeutic applications. Mention will inparticular be made of cell therapy for cancers (Fong L. and Engleman E.,Annu., Rev. Immunol., 2000; 18:245-273).

A major obstacle in the development of the various applications ofplasmacytoid dendritic cells is the problem of isolating and purifyingsufficient amounts of cells. Specifically, in humans, plasmacytoiddendritic cells (pDCs) represent less than 0.5% of the circulatingcells, which makes it very difficult to isolate them from peripheralblood in large amounts. Moreover, the isolated cells do not proliferatein vitro and die rapidly in culture. Thus, no line of immortalizedplasmacytoid dendritic cells (pDCs) is available at the current time.Only a dendritic cell line of myeloid and murine origin has beendescribed (Winzler C. et al., J. Exp. Med., 1997;l85:317-328).

Chaperot et al. (Chaperot L. et al., Blood., 2001;97:3210-3217) haveidentified a new leukemia entity, involving a tumor-related equivalentof plasmacytoid dendritic cells. These cells were identified duringinvestigations regarding CD4+ CD56+ leukemia tumor cells. In fact, thesecells could not be classified in any of the leukemia categoriesdescribed up until then. Chaperot et al. (Chaperot L. et al., Blood.,2001;97:3210-3217) put forward the hypothesis that these cells couldbelong to the pDC line. In fact, apart from CD56, the known phenotype oftumor cells could be superimposed on that of pDCs, in particular: CD4+,CD11c−, HLA-DR+, CD123+, CD45RA+. A functional study made it possible toshow that, as for normal pDCs, IL3 and GM-CSF promote survival of thetumor cells in vitro. In addition, these tumor cells express mRNAsencoding the chains preTalpha and lambda like 14.1 and, in the presenceof viruses, they are capable of secreting interferon-alpha. Furthermore,when the tumor cells are cultured in the presence of IL3, they undergo avery clear maturation, with a substantial increase in particular in theexpression of the costimulatory molecules CD40, CD80 and CD86, and alsothe appearance of CD1a, CD1c and CD83. This maturation is accompanied bythe acquisition of the ability to activate naïve T lymphocytes, whichare then oriented toward a Th2-type cytokine production profile. Theidentification of leukemia-related pDCs offers new perspectives withregard to the phenotypic and functional characterization of normal pDCs.Specifically, they represent a source of cells that have the samecharacteristics as normal pDCs. However, the pathology remains rare, 23patients having been identified by the GEIL: group d'étudesimmunologique des leucémies [leukemia immunology study group] (FeuillardJ. et al., Blood., 2002;99:1556-1563), and the recovery of large amountsof cells of quality is very difficult. In addition, the pDC cells takenfrom these patients do not naturally multiply in vitro, and methods forplacing the cells in culture and for culturing them, in vitro, have notbeen described.

The aim of the present invention is therefore to propose an in vitromethod for isolating a cell line of human plasmacytoid dendritic cells(pDCs). Any cell line thus obtained has many applications, in particularin the therapeutic field and in fundamental immunology.

DESCRIPTION OF THE INVENTION

The present invention relates to human plasmacytoid dendritic cell linesof phenotype CD4+, HLA-DR+, CD123+, CD45RA+, CD11c− and CD13−, and tothe method for obtaining them.

The term “cell line” applies to mammalian cells cultured in vitro.Primary cultures of mammalian cells do not multiply in culture or ceaseto multiply in culture after a limited number of divisions. The celllines according to the present invention are capable of multiplyingindefinitely, something of which primary or secondary cultures ofmammalian cells are incapable. These properties of the humanplasmacytoid dendritic cell (pDC) lines according to the invention makeit possible to advantageously obtain large amounts of cells bymultiplication or proliferation of these cells in vitro. Preferably, thecell lines according to the invention are isolated after at least 20,30, 40, 50, 60, 70, 80, 90, and preferably at least 100, cell divisions.

The plasmacytoid dendritic cell lines obtained according to theinvention can therefore be described as immortal since they multiplyindefinitely in vitro.

Plasmacytoid dendritic cells per se are known to those skilled in theart and can be identified by means of their morphologicalcharacteristics and by means of their surface phenotype. Thesemorphological characteristics are an extended, endoplasmicreticulum-rich and therefore basophilic cytoplasm with an excentricnucleus which makes them resemble plasma cells (Siegal, Science, 1999,284:1835-1837, Grouard G. et al., J. Exp. Med., 1997). Theseplasmacytoid dendritic cell (pDC) lines are also characterized by meansof their specific surface phenotype and in particular by means of thereceptors/antigens expressed at the surface of these cells. Thus, thedistinction of the various classes of immune system cells according tothe receptors/antigens (cell markers) expressed at the surface of thecells is a technique that is widely described in the literature. Thesesurface phenotype analyses are usually carried out by flow cytometry.Human plasmacytoid dendritic cells thus in particular express the CD4,HLA-DR, CD123 and CD45RA antigens. They lack the CD11c and CD13 markersspecific for myeloid dendritic cells. The methods for identifying cellshaving this phenotype have in particular been described by M. Cella etal. (Nat. Med., 1999; 5:919-923).

The term “plasmacytoid dendritic cells” is therefore intended to meancells that have the required morphological characteristics and a CD4+,HLA-DR+, CD123+, CD45RA+, CD11c−, CD13− phenotype.

Plasmacytoid dendritic cells characteristically express the BDCA2 andBDCA4 cell markers. The cells therefore have a BDCA2+, BDCA4+ phenotype.

A subject of the present invention is an in vitro method for isolating ahuman plasmacytoid dendritic cell line, comprising the steps consistingin:

-   -   a) culturing adherent stromal cells;    -   b) placing in culture leukemia tumor cells from a patient        suffering from plasmacytoid dendritic cell leukemia, on said        adherent stromal cells in a suitable culture medium; and    -   c) multiplying the cells by means of successive cell divisions        on said adherent stromal cells in a suitable culture medium, so        as to obtain a human plasmacytoid dendritic cell line.

A leukemia entity involving a tumor counterpart of human plasmacytoiddendritic cells has been described by Chaperot et al. (Blood,2001;97:3210-3217). Preferably, the leukemia tumor cells placed inculture have a CD4+, HLA-DR+, CD123+, CD45RA+, CD11c−, CD13− phenotype.Preferably, these tumor cells also have a CD56+ phenotype. Thisphenotype can be detected according to known methods of the state of theart.

Preferably, the isolation of the line is carried out by placing inculture and culturing on adherent stromal cells of the MS-5 murine line.

Preferably, the steps consisting in placing leukemia tumor cells inculture and multiplying the cells by means of successive cell divisionsso as to obtain a human plasmacytoid dendritic cell line are carried outin the presence of cytokines that support the proliferation andamplification of human hematopoietic cells.

Various cytokines that support the proliferation and amplification ofhuman hematopoietic progenitors and cells can be used in the methods forisolating a plasmacytoid dendritic cell (pDC) line according to theinvention. The amounts of cytokines to be used in the methods accordingto the invention are those that are conventionally used for cellcultures in vitro.

Preferably, the cells are placed in culture and multiplied in thepresence of at least one cytokine chosen from the group comprising IL6,FLT3-L, SCF, IL3, IL7, G-CSF and GM-CSF. More preferably, the cells areplaced in culture and multiplied in the presence of at least onecytokine chosen from the group comprising SCF and the FLT3-ligand.

Preferably, the cell lines according to the invention are establishedand isolated after at least 20, 30, 40, 50, 60, 70, 80, 90, andpreferably at least 100, cell divisions.

In a particular embodiment of the invention, the in vitro method forisolating a human plasmacytoid dendritic cell line comprises anadditional step (d) consisting in cloning the human plasmacytoiddendritic cell line obtained in step (c), so as to obtain various humanplasmacytoid dendritic cell lines or “clones”.

The “cloning of a line” denotes the individualization of cells of thisline, and the culturing and multiplying of the individualized cells soas to obtain cell clones or lines. The term “clone” is intended to meana collection of genetically identical cells obtained from a single cell.

In a particular embodiment, the in vitro method for isolating a humanplasmacytoid dendritic cell line therefore comprises an additional step(e) consisting in selecting said various human plasmacytoid dendriticcell lines or clones, so as to identify the clones having a phenotype ofinterest. According to a criterion of interest, the selection ofvariants having a particular characteristic may, for example, make itpossible to study the role of a targeted molecule in the function ofpDCs.

Among the various clones obtained from the multiplication of one cell,the clones of plasmacytoid human dendritic cells having a CD56+ or CD56−phenotype are thus, for example, selected.

Another subject of the present invention is an isolated cell line thatcan be obtained according to a method of the invention.

In a preferred embodiment, a subject of the invention is the humanplasmacytoid dendritic cell line, called GEN2.2, deposited with the CNCM(Collection Nationale de Cultures de Microorganismes [NationalCollection of Cultures of Microorganisms], Pasteur Institute, 25 rue duDocteur Roux, F-75015 Paris) on Sep. 24, 2002, under the CNCM numberI-2938 according to Rule 6.1 of the Treaty of Budapest, or a humanplasmacytoid dendritic cell line, called GEN 3, deposited on Oct. 16,2003, under the CNCM number I-3110 according to Rule 6.1 of the Treatyof Budapest.

Another subject of the present invention is a method for culturing andmultiplying, in vitro, a human plasmacytoid dendritic cell line,comprising the steps consisting in:

-   -   a) culturing adherent stromal cells; and    -   b) culturing the human plasmacytoid dendritic cell line on said        adherent stromal cells in a suitable culture medium.

The human plasmacytoid dendritic cell lines according to the inventionare capable of multiplying indefinitely when they are cultured onadherent stromal cells. Various adherent stromal cell lines are known tothose skilled in the art.

In the usual manner, the cells are cultured in plastic flasks,containers and dishes commonly used in this field, that allow the cellsto adhere to the solid support. Advantageously, plastic flasks,containers and dishes are “precoated” or pre-seeded with adherentstromal cells.

In a particular embodiment, the stromal cells are cultured untilconfluency.

In an advantageous embodiment, the stromal cells are then irradiated inorder to stop their proliferation, before beginning the culturing of thehuman plasmacytoid dendritic cell lines on said stromal cells.

The stromal cells are known to those skilled in the art. They aretypically animal or human stromal cells derived from bone marrow or fromother organs. Adherent stromal cells capable of supporting theproliferation of human progenitor cells are preferably used.

The adherent stromal cells are preferably chosen from the groupcomprising S17 stromal cells, AFT024 or M2-10B4 stromal cells (ATCC CRL1972), HESS-5 stromal cells and MS-5 stromal cells (Itoh et al., Exp.Hematol., 1989; 17:143-147).

Advantageously, the adherent stromal cells are adherent stromal cells ofthe MS-5 murine line (deposited with the DSMZ [German Collection ofMicroorganisms and Cell Cultures] under the No. ACC441).

Culture media for culturing mammalian cell lines in vitro are well knownto those skilled in the art and commonly used. Preferably, the usualculture media, such as RPMI 1640 Glutamax (GIBCO® culture media)supplemented with sodium pyruvate, nonessential amino acids anddecomplemented fetal calf serum, are used.

Another subject of the present invention is an in vitro method forobtaining activated human plasmacytoid dendritic cells, characterized inthat it comprises the following steps:

-   -   a) an isolated cell line according to the invention is provided;    -   b) said cell line of step a) is activated so as to obtain        activated human plasmacytoid dendritic cells.

Preferably, said cell line is activated with a virus and/or IL3 and/orCD40.

The methods for activating, in vitro, a human plasmacytoid dendriticcell line according to the invention make it possible to obtain a largenumber of activated or mature plasmacytoid dendritic cells. The presentinvention also relates to activated or mature human plasmacytoiddendritic cells obtained from the cell lines according to the invention.

Methods for activating plasmacytoid dendritic cells are known to thoseskilled in the art (Grouard G. et al., J. Exp. Med., 1997; Cella M. etal., Nat. Immunol., 2000;1:305-310). The activation or the maturation ofthe human plasmacytoid dendritic cell lines is therefore carried outaccording to usual techniques. Unlike the methods of the state of theart, the methods according to the present invention make it possible toobtain a large number of activated or mature cells by virtue of the useof human plasmacytoid dendritic cell lines according to the invention.

According to a first embodiment of the invention, the cell lines areactivated with an enveloped or naked, single-stranded ordouble-stranded, RNA virus (for example, HIV, HTLV, influenza, mumps,measles, dengue and ebola) or DNA virus (for example, adenovirus, HSV,CMV, EBV), or derivatives thereof (poly-IC), with bacteria (for example,M. tuberculosis) or derivatives thereof (CpG ODN), or with parasites(for example, leishmania) or fungi (for example, Candida albicans)

Preferably, the activation is carried out in the presence of at leastone virus chosen from influenza, HIV and HSV.

According to a second embodiment, the human plasmacytoid dendritic celllines according to the invention are activated with stimuli of Tlymphocyte origin, which are soluble factors such as cytokines (forexample, IL3, GM-CSF or IFNα) or ligands that interact with surfacereceptors such as the proteins of the TNF family (CD40L or anti-CD40,for example).

Preferably, the human plasmacytoid dendritic cell lines according to theinvention are activated with IL3 and/or CD40L.

According to a particularly advantageous embodiment, the humanplasmacytoid dendritic cell lines according to the invention areactivated with IL3, CD40L and a virus.

By way of example, the activation or the maturation of the plasmacytoiddendritic cell lines is induced by the addition of virus, of IL3-CD40Lor of virus-IL3-CD40L to the culture medium.

The present invention also relates to any isolated, activated humanplasmacytoid dendritic cell line that can be obtained according to anymethod described above.

Activated or mature human plasmacytoid dendritic cells are known tothose skilled in the art and can be identified or detected according tousual techniques.

Typically, activated or mature human plasmacytoid dendritic cellssecrete at least one molecule chosen from pro-inflammatory cytokines(for example, IL6, TNFα and IFNα), cytokines that orient the immuneresponse (for example, IL12 and IFNα), chemokines (for example, IL-8,RANTES, IP10, MIG, MDC, TARC, I309) and antiviral cytokines (forexample, IFNα).

Preferably, the activated (or mature) human plasmacytoid dendritic cellsderived from cell lines according to the invention secrete at least onemolecule chosen from IL12, TNF, IL6, IL8 and IFNα.

Conventionally, activated or mature human plasmacytoid dendritic cellsare also characterized in morphological terms by means of numerousdendrites and by means of their phenotype. The maturation or theactivation of the cells is thus accompanied by a large increase in theexpression of HLA molecules of costimulatory molecules (for example,CD40, CD80, CD83, CD86) and of chemokine receptors (for example, CCR6and CCR7). These cells then become capable of activating naïve Tlymphocytes (Grouard G. et al., J. Exp. Med., 1997, Cella M. et al.,Nat. Immunol., 2000;1:305-310, Rissoan M C. et al., Science.,1999;283:1183-1186).

Preferably, the activated (or mature) human plasmacytoid dendritic cellsderived from cell lines according to the invention express at least onecell marker chosen from HLA I, CD86, CD80, CCR6, CCR7 and CD83.

The cells derived from the activation or from the maturation of a cellline of plasmacytoid dendritic cells according to the invention exhibitsat least one of the characteristics (secretion, morphology, phenotype,ability to activate naïve T lymphocytes) of activated or matureplasmacytoid dendritic cells.

The present invention also relates to a method for activating Tlymphocytes, in vitro, characterized in that it comprises the followingsteps:

-   -   a) an isolated cell line according to the invention is provided;    -   b) said cell line of step a) is activated so as to obtain        activated human plasmacytoid dendritic cells;    -   c) T lymphocytes are brought into contact with said activated        human plasmacytoid dendritic cells of step b).

Preferably, the cell line of plasmacytoid dendritic cells is activatedwith a virus, IL3 and/or CD40.

This method can be carried out on any type of biological samplecomprising T lymphocytes. Preferably, it is a human or animal biologicalsample. The sample is preferably blood and, for applications of theimmunotherapy and cell therapy type, it is autologous blood.

The present invention also relates to a method for identifying compoundsthat activate human plasmacytoid dendritic cells, comprising the stepsconsisting in:

-   -   a) bringing the compound into contact with the plasmacytoid        dendritic cell line according to the invention;    -   b) detecting the activation of said cell line.

The detection of the activation or of the maturation of the humanplasmacytoid dendritic cells is carried out according to conventionaltechniques known to those skilled in the art.

In one embodiment, the secretion of at least one molecule chosen frompro-inflammatory cytokines (for example, IL6, TNFα and IFNα), cytokinesthat orient the immune response (for example, IL12 and IFNα), chemokines(for example, IL-8, RANTES, IP10, MIG, MDC, TARC, I309) and antiviralcytokines (for example, IFNα) is detected.

Preferably, the secretion of at least one molecule chosen from IL12,TNF, IL6, IL8 and IFNα is detected.

In another embodiment, the increase in expression of HLA molecules, ofcostimulatory molecules (for example, CD40, CD80, CD86), of CD83 and ofchemokine receptors (for example, CCR6 and CCR7) is detected.

The present invention also relates to a pharmaceutical compositioncomprising at least one cell of a plasmacytoid dendritic cell lineaccording to the invention.

The plasmacytoid dendritic cell lines according to the invention are oftherapeutic interest in the field of the antimicrobial and antitumorimmune response.

The plasmacytoid dendritic cell lines according to the invention can beused in the treatment of various types of pathologies, in particular inthe treatment of pathologies associated with infectious or microbialagents (bacteria, viruses, parasites, fungi), of cancers, of allergiesand of autoimmune diseases.

More particularly, the plasmacytoid dendritic cells are capable ofpresenting tumor or microbial antigens in in vitro or ex vivo systems soas to induce an immune response against the tumor cells or the infectedcells.

The present invention relates more particularly to the field ofantitumor immunotherapy and cell therapy. The plasmacytoid dendriticcell lines according to the invention can thus be used as animmunotherapy agent.

The dendritic cell lines according to the invention are also used forproducing a pharmaceutical composition, and advantageously for producinga composition capable of promoting an antitumor immune response for thetreatment of cancers or an antimicrobial response for the treatment ofinfectious diseases.

The examples and figures below will make it possible to demonstratecertain advantages and characteristics of the present invention.

DESCRIPTION OF FIGURES

FIG. 1: Proliferation of the GEN2.2 line

After the first 35 days of culture, a regular proliferation rate wasobtained.

Each week, 0.6 million GEN2.2 cells were co-seeded with cells of theMS-5 line, and then diluted after 3 days. Two counts per week wereperformed. The theoretical cumulative number of cells was calculatedfrom these counts.

FIG. 2: Cloning of the GEN2.2 line

The 22 subclones of the GEN2.2 line obtained by limiting dilutionexpress variable levels of CD56 and BDCA2 (flow cytometry measurement ofthe mean fluorescence or of the percentage of positive cells) (A). Nocorrelation exists between the levels of expression of these twomarkers. Among the clones studied, 1 was negative for CD56 expression(3B9), 16 were heterogeneous (for example: 3A9), and 5 were verypositive (for example: 1B10) (B).

FIG. 3: Maturation of the cells of the GEN2.2 line

The cells of the GEN2.2 line were placed in culture for 48 h in thepresence of IL3+CD40L, of influenza virus or of the three signals, andthen phenotyped by flow cytometry. Under the three conditions, a clearmaturation of the cells was observed, reflected by an increase inexpression of the molecules associated with antigen-presenting functions(HLA I, CD40, CD80, CD86), and other modifications (increase in CCR6 andCCR7, decrease in CXCR4 and BDCA2).

FIG. 4: Activation and Th1/Th2 polarization of naïve T lymphocytes

After 48 h of culture in the presence of IL3+CD40L, of virus or of thethree signals, the cells of the GEN2.2 line were capable of inducing theproliferation of naïve CD4+ T lymphocytes (A).

The proliferation was measured by incorporation of triturated thymidineover 18 h, at the end of 6 days of mixed culture. The T lymphocytesactivated in the course of this MLR express CCR4 if they have beenactivated with GEN2.2 cells preactivated in the presence of IL3+CD40L,whereas they express CCR5 and CXCR3 when they have been activated withcells of the GEN2.2 line preactivated in the presence of virus orvirus+IL3+CD40L (B). The supernatants of the mixed culture and of the Tlymphocytes reactivated at the end of the MLR with PMA+ionomycin exhibitmore IFNg when the T cells have been activated with cells of the GEN2.2line preactivated in the presence of virus or virus+IL3+CD40L, whereasmore IL4 or more IL5 is found when the T cells have been activated withcells of the GEN2.2 line preactivated in the presence of IL3+CD40L(assaying by means of the CBA (Becton Dickinson) cytometry technique)(C). The detection of the percentage of cells secreting IFNg or IL4 wascarried out after activation, with PMA+ionomycin, of the T lymphocytesat the end of the MLR. Fixed and permeabilized cells are labeled withantibodies specific for these cytokines and passed through the cytometer(D).

EXAMPLES Example 1 Generation of a Human Line of Plasmacytoid DendriticCells

The leukemia pDC cells were isolated from a sample of peripheral bloodfrom the patient GEN previously described (Chaperot L. et al., Blood,2001;97:3210-3217). The isolated mononuclear cells that were frozencontained more than 98% of tumor cells. The MS-5 murine adherent stromalline (Bennaceur-Griscelli A. et al., Blood, 1999;94:529-538; Issaad C.et al., Blood, 1993;81:2916-2924) was used as a “feeder”. Five milliontumor cells were placed in culture in a flask pre-coated with cells ofthe MS-5 line at confluency, in 5 ml of complete medium (RPMI 1640Glutamax (GIBCO® culture media), supplemented with 1 mM of sodiumpyruvate, 100 U/ml of penicillin, 100 μg/ml of streptomycin,nonessential amino acids, and 10% of decomplemented fetal calf serum).For the first five weeks of the culture, SCF (Nishi N. et al., Exp.Hematol., 1996;24:1312-1321) (stem cell factor) and Flt3-ligand(Pulendran B. et al., J. Immunol., 2000;165:566-572; Blom B. et al., J.Exp. Med., 2000;192:1785-1796) were added. The cells were counted anddiluted with fresh medium containing the cytokines each week. At thisstage of the culture, regular proliferation was obtained, and during thefollowing four weeks, 0.6 million cells (then called GEN2) wereco-seeded each week in a new flask with 0.6 million MS-5. The SCF andthe Flt3-L were then eliminated, and the line continued to proliferatesatisfactorily on the MS-5 sublayer; it was then called GEN2.2. Thisline could be maintained for at least 5 months in culture (FIG. 1). Thedoubling time of the line is 1.1 days. Irradiation of the MS-5 sublayer(60Gy) makes it possible to support in an identical manner theproliferation of the GEN2.2 line, while at the same time eliminating thecontaminating MS-5 cells that could disturb certain functional analysesdue to their proliferation. In the absence of MS-5, the GEN2.2 cellscease to proliferate and die.

Example 2 Phenotypic and Karyotypic Analysis of the GEN2.2 Line

The phenotype of the cells of the GEN2.2 line is determined by flowcytometry, using antibodies directly labeled with FITC or PE.

Like normal pDCs, the fresh cells of the GEN2.2 line are characterizedby their expression of CD4, HLA ABC, HLA DR, CD45RA, CD123 (IL3-Ra),ILT-3, BDCA2 and BDCA4 (Table 1). CD7 is present, and 50% of the cellsexpress CD56, whereas the other pan-T, B and NK markers are not detected(CD3, CD8, CD19, CD20, CD16, CD57). The myeloid markers (CD13, CD11b,CD11c, CD14, CD64) are negative, whereas CD33 is positive. As regardsthe costimulatory molecules, the GEN2.2 cells express CD86, 27% arepositive for CD40, whereas CD80 is absent. CD1a, CD1c and CD83 are notdetected. The chemokine/homing receptors such as CCR1, CCR2, CCR3, CCR4,CCR5, CXCR1, CXCR2, CXCR5 and CLA are not expressed. CCR6 and CCR7 arefound on 10 to 15% of the cells. More than 40% of the cells of theGEN2.2 line express CXCR3, CXCR4 and CD62L.

TABLE 1 GEN2.2 cell phenotype T/B celis CD3 CD4 CD7 CD8 CD19 CD20 0 10095 2 0 0 Myeloid/monocyte CD13 CD33 CD11b CD11c CD14 CD64 CD116 2 66 210 0 1 25 Natural killer cells CD16 CD56 CD57 0 51 0 Dendritic cellsCD1a CD1c CD83 BDCA2 BDCA4 CD123 ILT-3 4 5 2 70 88 100 100 Antigenpresenting cells CD40 CD80 CD86 HLA ABC HLA DR 27 2 100 100 100Misceallaneous CD34 CD36 CD38 CD45RA CD45RO CD65 CD117 0 89 74 90 21 170 Chemokine/homing receptors CCR1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 0 1 5 26 8 16 CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CLA CD62L 0 6 44 73 1 0 94

Among the 11 mitoses analyzed, 7 had the following karyotype49,XY,+6,t(6:8)(p21;q24),+r(12),+20, and 4 had 49,iden,t(3;5)(q21;q21).This karyotype is identical to that of two subclones present in theinitial tumor cells (patient UPN24) (Leroux D. et al., Blood,2002;99:4154-4159).

Example 3 Subcloning of the GEN2.2 Line

A suspension of GEN2.2 cells was placed in culture on a confluent MS-5sublayer, in flat-bottomed 96-well plates, in 0.2 ml of complete medium.0.3 cell/well was deposited into two plates (called 1 and 2) and 1cell/well was deposited into one plate (called 3). After 2 to 5 weeks ofculture, the proliferating wells were transferred into 24-well plates,and then into culture flasks, always on an MS-5 sublayer.

19/96 wells grew in plate 3, and 12/192 grew in plates 1 and 2. 22 ofthese clones (10 of plates 1 and 2, and 12 of plate 3) were amplified,phenotyped and frozen. 5 of these clones strongly expressed the CD56molecule (more than 99% of positive cells, with an MFI (meanfluorescence intensity) >1000); 1 clone does not express CD56 (less than10% of positive cells, MFI<10); 16 clones exhibit heterogeneous CD56expression (49 to 98% of positive cells, 33<MFI<800) (FIG. 2). All theclones are positive for HLA DR and CD4, and behave in the same way asthe GEN2.2 line in terms of growth.

Example 4 Induction of the Maturation of GEN2.2 Cells with the InfluenzaVirus and/or IL3+CD40L

The cells were cultured in complete medium, in the absence of MS-5, at0.5 million/ml. Three culture conditions were evaluated:

“virus”, in the presence of influenza virus (strain A/NewCaledonia/20/99, subtype H1N1, Aventis Pasteur, 137×10⁻³ μg ofhemagglutinin/ml),

“IL3-CD40L”, by addition of IL3 (10 ng/ml) and of recombinant solubleCD40L (1 μg/ml, Alexis),

“virus-IL3-CD40L”.

After 48 h of culture, the culture supernatants were frozen for assayingoff IFNα by ELISA (Beckman Coulter), and of ILL-β, IL2, IL4, IL5, IL-6,IL-8, IL-10, IL-12p70, IFN and TNF using the TH1/TH2 and inflammatorycytokine “cytometric bead array” (CBA, BD Bioscience) kits. The cellswere recovered for phenotyping by cytometry, and cytospin smears withMGG staining.

As shown in FIG. 3, the cells of the GEN2.2 line mature when they areplaced in culture in the presence of IL3-CD40L, of virus, or of thethree signals. This maturation is reflected by a very clear increase inlevels of expression of HLA I, CD86 and CD80 molecules, that areassociated with antigen-presenting functions. The HLA-DR molecules alsoincrease, but to a lesser extent. The expression of CD40 and CD83 isespecially increased under the two conditions where the virus ispresent. The GEN2.2 cells also acquire CD1a and CD1c molecules, whichare known to be expressed by myeloid-type DCs. Moreover, they looseBDCA2 under all these conditions, and also CXCR4. The expression ofBDCA2 is in fact associated with an immature stage of DCs, allowing themto take up viruses (Dzionek A. et al., J. Exp. Med., 2001;194:1823-1834)and involved in the regulation of IFNα secretion. The loss of CXCR4, theligand of which is SDF1, a molecule present in the secondary lymphoidorgans, could suggest that the pDCs do not follow the same circulatorypathways as the myeloid DCs in returning to the lymph nodes; in fact,MDCs express CXCR4 only at the mature stage (Sallusto F. et al., Eur. J.Immunol., 1998;28:2760-2769). During their maturation, the GEN2.2 cellsacquire CCR7, which could allow them to migrate to the secondarylymphoid organs where they could encounter the naïve T lymphocytes inorder to activate them, this up-regulation also being described on DCsof myeloid origin (Sallusto F. et al., Eur. J. Immunol.,1998;28:2760-2769). The inversion of the CD45RA/RO ratio observed duringthe maturation of the cells, and also the acquisition of CCR6, are datathat are not yet described on normal pDCs.

The supernatants of the cells activated with the virus contain TNFα,IL6, IL8 and IFNα. The secretion of these cytokines is zero or very lowunder the “IL3-CD40L” condition, but when the three signals arecombined, the amount of TNFα, of IL6 and of IL8 detected is multipliedthree-fold compared with the virus condition (Table 2), whereas the IFNαis unchanged, or slightly decreased. Entirely advantageously, IL12p70 ispresent under the “virus-IL3-CD40L” condition. This cytokine plays avery important role in the activation of Th1 lymphocytes and thedifferentiation of cytotoxic T lymphocytes that are fundamental incombating viruses. The production of IL12 by normal pDCs has beendescribed by certain authors (Cella M. et al., Nat. Med.,1999;5:919-923; Cella M. et al., Nat. Immunol., 2000;1:305-310; Krug A.et al., Eur. J. Immunol., 2001;31:3026-3037), whereas many other studieshave rather described their inability to produce this cytokine (RissoanM C et al., Science., 1999;283:1183-1186; Ito T. et al., J. Exp. Med.,2002;195:1507-1512; Gilliet M. and Liu Y J., J. Exp. Med.,2002;195:695-704; Kadowaki N. et al., J. Exp. Med., 2001;194:863-869;Bauer M. et al., J. Immunol., 2001;166:5000-5007). We detected none ofthe other cytokines tested in the supernatants of these cells.

TABLE 2 Cytokines produced by the activated GEN2.2 cells IL12p70* TNF*IL6* IL8* IFNα⁺ IL3-CD40L 2 45 5 23 0 Virus 3 536 2391 391 1953Virus-IL3- 190 1776 8690 2580 1657 CD40L *pg/ml, assay by the CBAtechnique ⁺IU/ml, assay by ELISA

Example 5 Th1/Th2 Polarization of Naïve T Lymphocytes

The cells of the GEN2.2 line were preactivated under the threeconditions described in Example 4, in order to evaluate their ability tostimulate the proliferation of naïve T lymphocytes and to induce theirdifferentiation along the Th1 or Th2 pathway. Naïve CD4+ T lymphocyteswere purified from cord blood mononuclear cells using an immunomagnetictechnique (stem cell technology). To measure the proliferation, 25 000naïve T lymphocytes per well were stimulated with 25 000, 10 000, 5000,1000 and 250 preactivated cells of the GEN2.2 line. Triturated thymidineincorporation was measured on the 6th day, over the last 18 hours of theculture. To evaluate the Th1/Th2 polarization, 50 000 naïve Tlymphocytes per well were stimulated with 10 000 preactivated cells ofthe GEN2.2 line. After 6 days of culture, the culture supernatants werefrozen and the recovered cells were phenotyped (CCR4, CCR5, CXCR3), andactivated with phorbol myristate acetate (PMA 5 ng/ml)+ionomycin (0.5μg/ml) for 4 hours or 6 hours in the presence of monensin (3 μM, Sigma)for the last 4 hours. IL2, IL4, IL5, IL10, TNFα and IFNγ were assayed inthe supernatants, before and after activation with PMA+ionomycin for 4h, using the Th1/Th2 CBA kit. The cells activated with PMA+ionomycin for6 h in the presence of the secretion inhibitor were fixed (FACS Lysingsolution, Becton Dickinson), and then permeabilized (FACS Permeabilizingsolution, Becton Dickinson). The presence of Th1 or Th2 cells wasdetected by cytometry, with intracytoplasmic labeling using anti-IFNγand anti-IL4 antibodies.

The greatest proliferation (25 000 cpm) of naïve CD4+ T cells wasobtained with the cells preactivated under the “virus-IL3-CD40L”condition; the “IL3-CD40L” and “virus” preactivated cells are, however,capable of inducing naïve T cell proliferation (10 000 cpm), confirmingthe dendritic cell-type APC potentialities of the cells of the GEN2.2line (FIG. 4A). In fact, only dendritic cells are capable of effectivelyactivating naïve T cells. The polarization of the lymphocytes thusactivated along the Th1 or Th2 pathways was evaluated according toseveral criteria. First of all, the expression of CCR4, described asbeing associated with Th2 cells, is higher at the surface of the T cellsactivated with the “IL3-CD40L” cells, whereas the expression of CCR5 andCXCR3, described with regard to Th1 cells, is greater on the T cellsactivated with the “virus” and “virus-IL3-CD40L” cells (Sallusto F. etal., Immunol. Today., 1998;19:568-574) (FIG. 4B). Analysis of thecytokines in the culture supernatants shows that IL5 and IL4 (Th2cytokines) are produced under the conditions where the T cells have beenactivated with the “IL3-CD40L” and “virus-IL3-CD40L” cells, whereas IFNγ(Th1) is especially detected under the “virus” and “virus-IL3-CD40L”conditions. IL10 is found in all the cases, possibly suggesting thepresence of regulatory lymphocytes (Levings M K. et al., J. Exp. Med.,2001;193:1295-1302; Dieckmann D. et al., J. Exp. Med.,2001;193:1303-1310) (FIG. 4C). Confirming these results, the detectionof intracytoplasmic cytokines shows a greater percentage ofIFNγ-producing cells among the T cells activated with the “virus” and“virus-IL3-CD40L” cells, and a lower percentage of IL4-producing cellsunder the “virus” condition (FIG. 4D).

These results therefore show a preferential orientation along the Th2pathway for the naïve T cells activated with the “IL3-CD40L” GEN2.2cells, whereas a profile that is rather Th1 is induced with the “virus”cells, as has been described for normal pDCs (Rissoan M C. et al.,Science, 1999;283:1183-1186; Cella M. et al., Nat. Immunol.,2000;1:305-310; Kadowaki N. et al., J. Exp. Med., 2000;192:219-226). The“virus-IL3-CD40L” condition induces the strongest activation of Tlymphocytes, orientating them rather toward a Th1 profile (CCR5+,CXCR3+, very strong production of IFNγ), with, however, the paralleldifferentiation of Th2 cells (production of IL4 and of IL5).

Example 6 GEN 3 Cell Line

The leukemia pDC cells were isolated from a sample of peripheral bloodfrom the patient GEN previously described (Chaperot L. et al., Blood.,2001;97:3210-3217). The isolated mononuclear cells that were frozencontain more than 98% of tumor cells. The MS-5 murine adherent stromalline (Bennaceur-Griscelli A. et al., Blood., 1999;94:529-538; Issaad C.et al., Blood., 1993; 81:2916-2924) was used as a “feeder”; it is usedafter having been irradiated at 60 Gy. One million tumor cells wereplaced in culture in a flask precoated with 1 million cells of the MS-5line, in 5 ml of complete medium (RPMI 1640 Glutamax (GIBCO® culturemedia), supplemented with 1 mM of sodium pyruvate, 100 U/ml ofpenicillin, 100 μg/ml of streptomycin, nonessential amino acids, and 10%of decomplemented fetal calf serum). The cells were counted and dilutedwith fresh medium each week, and regular proliferation was obtained.This line could be maintained for at least 3 months in culture. The linewas deposited with the CNCM on 10/16/2003, under the number I-3110.

1. A human immortal plasmacytoid dendritic cell line called GEN2.2,deposited with the CNCM under the CNCM number I-2938.
 2. A humanimmortal plasmacytoid dendritic cell line called GEN3, deposited withthe CNCM under the CNCM number I-3110.
 3. A method for obtainingactivated human plasmacytoid dendritic cells, in vitro, comprising: a)providing cells as claimed in claim 1; and b) activating the cells ofstep a) with viruses, parasites, fungi and/or stimuli of T lymphocyteorigin to obtain activated human plasmacytoid dendritic cells.
 4. Themethod as claimed in claim 3, characterized in that the cells areactivated with a virus and/or IL3 and/or CD40.
 5. A method foractivating T lymphocytes, in vitro, comprising: a) providing cells asclaimed in claim 1; b) activating the cells of step a) with viruses,bacteria, CpG-ODN, parasites, fungi and/or stimuli of T lymphocyteorigin to obtain activated human plasmacytoid dendritic cells; and c)bringing T lymphocytes into contact with said activated humanplasmacytoid dendritic cells of step b) to obtain activated Tlymphocytes.
 6. The method for activating T lymphocytes, in vitro, asclaimed in claim 5, characterized in that, in step b), the cells areactivated with a virus and/or IL3 and/or CD40.
 7. A method foridentifying compounds that activate human plasmacytoid dendritic cells,comprising: a) bringing a compound into contact with cells as claimed inclaim 1; and b) detecting whether the cells are activated.
 8. A methodfor obtaining activated human plasmacytoid dendritic cells, in vitro,comprising: a) providing cells as claimed in claim 2; and b) activatingthe cells of step a) with viruses, parasites, fungi and/or stimuli of Tlymphocyte origin to obtain activated human plasmacytoid dendriticcells.
 9. A method for activating T lymphocytes, in vitro, comprising:a) providing cells as claimed in claim 2; b) activating the cells ofstep a) with viruses, bacteria, CpG-ODN, parasites, fungi and/or stimuliof T lymphocyte origin to obtain activated human plasmacytoid dendriticcells; and c) bringing T lymphocytes into contact with said activatedhuman plasmacytoid dendritic cells of step b) to obtain activated Tlymphocytes.
 10. A method for identifying compounds that activate humanplasmacytoid dendritic cells, comprising: a) bringing a compound intocontact with cells as claimed in claim 2; and b) detecting whether thecells are activated.